Elucidating Carfilzomib’s Induced Cardiotoxicity in an In Vivo Model of Aging: Prophylactic Potential of Metformin

Background: Carfilzomib is a first-line proteasome inhibitor indicated for relapsed/refractory multiple myeloma (MM), with its clinical use being hampered by cardiotoxic phenomena. We have previously established a translational model of carfilzomib cardiotoxicity in young adult mice, in which metformin emerged as a prophylactic therapy. Considering that MM is an elderly disease and that age is an independent risk factor for cardiotoxicity, herein, we sought to validate carfilzomib’s cardiotoxicity in an in vivo model of aging. Methods: Aged mice underwent the translational two- and four-dose protocols without and with metformin. Mice underwent echocardiography and were subsequently sacrificed for molecular analyses in the blood and cardiac tissue. Results: Carfilzomib decreased proteasomal activity both in PBMCs and myocardium in both protocols. Carfilzomib induced mild cardiotoxicity after two doses and more pronounced cardiomyopathy in the four-dose protocol, while metformin maintained cardiac function. Carfilzomib led to an increased Bip expression and decreased AMPKα phosphorylation, while metformin coadministration partially decreased Bip expression and induced AMPKα phosphorylation, leading to enhanced myocardial LC3B-dependent autophagy. Conclusion: Carfilzomib induced cardiotoxicity in aged mice, an effect significantly reversed by metformin. The latter possesses translational importance as it further supports the clinical use of metformin as a potent prophylactic therapy.

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Synergistic activity of the proteasome inhibitor PS-341 with non-myeloablative 153-Sm-EDTMP skeletally targeted radiotherapy in an orthotopic model of multiple myeloma

Blood ◽

10.1182/blood-2005-09-3870 ◽

2006 ◽

Vol 107 (10) ◽

pp. 4063-4070 ◽

Cited By ~ 25

Author(s):

Apollina Goel ◽

Angela Dispenzieri ◽

Susan M. Geyer ◽

Suzanne Greiner ◽

Kah-Whye Peng ◽

...

Keyword(s):

Multiple Myeloma ◽

Proteasome Inhibitor ◽

In Vivo Model ◽

Synergistic Activity ◽

Mineral Density ◽

Orthotopic Model ◽

Targeted Radiotherapy ◽

Myeloma Cells ◽

Cell Counts

Multiple myeloma is a highly radiosensitive skeletal malignancy, but bone-seeking radionuclides have not yet found their place in disease management. We previously reported that the proteasome inhibitor PS-341 selectively sensitizes myeloma cells to the lethal effects of ionizing radiation. To extend these observations to an in vivo model, we combined PS-341 with the bone-seeking radionuclide 153-Sm-EDTMP. In vitro clonogenic assays demonstrated synergistic killing of myeloma cells exposed to both PS-341 and 153-Sm-EDTMP. Using the orthotopic, syngeneic 5TGM1 myeloma model, the median survivals of mice treated with saline, 2 doses of PS-341 (0.5 mg/kg), or a single nonmyeloablative dose of 153-Sm-EDTMP (22.5 MBq) were 21, 22, and 28 days, respectively. In contrast, mice treated with combination therapy comprising 2 doses of PS-341 (0.5 mg/kg), 1 day prior to and 1 day following 153-Sm-EDTMP (22.5 MBq) showed a significantly prolonged median survival of 49 days (P < .001). In addition to prolonged survival, this treatment combination yielded reduced clonogenicity of bone marrow–resident 5TGM1 cells, reduced serum myeloma–associated paraprotein levels, and better preservation of bone mineral density. Myelosuppression, determined by peripheral blood cell counts and clonogenicity assays of hematopoietic progenitors, did not differ between animals treated with 153-Sm-EDTMP alone versus those treated with the combination of PS-341 plus 153-Sm-EDTMP. PS-341 is a potent, selective in vivo radiosensitizer that may substantially affect the efficacy of skeletal-targeted radiotherapy in multiple myeloma.

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Development of 3D Cerebral Aggregates in the Brain Ventricles of Adult Mice

Russian Journal of Developmental Biology ◽

10.1134/s1062360421030061 ◽

2021 ◽

Vol 52 (3) ◽

pp. 164-175

Author(s):

K. K. Sukhinich ◽

K. M. Shakirova ◽

E. B. Dashinimaev ◽

M. A. Aleksandrova

Keyword(s):

Blood Supply ◽

Three Dimensional ◽

In Vivo Model ◽

Cell Culture Process ◽

Specific Cell ◽

Cell Aggregates ◽

Lateral Ventricles ◽

Adult Mice ◽

The Brain

Abstract The cerebral organoids are three-dimensional cell cultures formed from brain-specific cell types arising from embryonic or pluripotent stem cells. Organoids provide an opportunity to study the early stages of brain development and diseases of the central nervous system. However, the modeling of organoids is associated with a number of unsolved problems. Organoid production techniques involve a complex cell culture process that requires special media, growth factors, and often the use of a bioreactor. Even under standardized conditions, structures of different morphology are formed: from disorganized cell aggregates to structured minibrains, which are selected for study. For natural reasons, organoids grown in vitro do not have a blood supply, which limits their development. We tried to obtain cerebral aggregates similar to organoids in an in vivo model, where vascular growth and tissue blood supply are provided, for which we transplanted a cell suspension from the mouse embryonic neocortex into the lateral ventricles of the brain of adult mice. Therefore, the medium for cultivation was the cerebrospinal fluid, and the lateral ventricles of the brain, where it circulates, served as a bioreactor. The results showed that the neocortex from E14.5 is a suitable source of stem/progenitor cells that self-assemble into three-dimensional aggregates and vascularized in vivo. The aggregates consisted of a central layer of mature neurons, the marginal zone free of cells and a glia limitans, which resembled cerebral organoids. Thus, the lateral ventricles of the adult mouse brain can be used to obtain vascularized cell aggregates resembling cerebral organoids.

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Preclinical Efficacy of the Investigational Orally Bioavailable Proteasome Inhibitor MLN9708 in Myeloma Bone Disease

Blood ◽

10.1182/blood.v120.21.4014.4014 ◽

2012 ◽

Vol 120 (21) ◽

pp. 4014-4014

Author(s):

Antonio Garcia-Gomez ◽

Dalia Quwaider ◽

Enrique M Ocio ◽

Laura San-Segundo ◽

Teresa Paíno ◽

...

Keyword(s):

Mouse Model ◽

Bone Formation ◽

Proteasome Inhibitor ◽

Serum Levels ◽

In Vivo Model ◽

Healthy Donors ◽

And Function ◽

Rpmi 8226

Abstract Abstract 4014 Introduction: Bone destruction, a hallmark of multiple myeloma (MM), arises as a consequence of the interactions between MM cells and the bone marrow microenvironment, which lead to an increase in the bone-resorptive activity and number of osteoclasts (OC) and a reduction of the bone-forming activity and differentiation of osteoblasts (OB). MLN9708, which hydrolyzes to pharmacologically active MLN2238 in aqueous solution, is an investigational proteasome inhibitor (PI) with demonstrated preclinical anti-myeloma activity. However, it is currently not known whether MLN9708, may have a beneficial effect on myeloma-associated bone disease. Here, we have conducted in vitro and in vivo studies to evaluate its ability to promote osteogenic differentiation and to inhibit OC formation and function in the myeloma setting. Patient samples, material and methods: The human MM cell lines RPMI-8226 and MM.1S (or RPMI-8226-luc and MM.1S-luc) together with the mesenchymal stem hMSC-TERT cell line were employed. Also, MSCs from BM samples of healthy donors and MM patients were used in OB differentiation studies, whereas PBMCs from healthy volunteers were used to generate OCs. NOD.SCID.IL2Rγ−/− mice were used in the in vivo model of disseminated human MM. MLN2238 and bortezomib (Velcade) were provided by Millennium Pharmaceuticals, Inc. OB differentiation from MSCs and OB function were investigated by measurement of ALP activity, quantitative mineralization, luciferase reporter assays, siRNA gene silencing and real time RT-PCR. The effect of the new PI on OC formation was assessed by enumeration of multinucleated (≥3) TRAP-positive cells. Measurement of resorbed area, immunofluorescence and flow cytometry were used to further investigate the effect of MLN2238 on OC function. In our in vivo model, bioluminescence imaging, micro-CT analysis and serum levels of Igλ and bone markers were determined. Results: Physiologic concentrations of MLN2238 were able to stimulate the osteogenic differentiation of MSCs from both myeloma patients and healthy donors in vitro to an extent comparable to bortezomib; this was assessed by increased levels of ALP activity, higher expression of bone formation markers (Runx2, osterix, osteopontin and osteocalcin) and augmented matrix mineralization. The enhanced OB formation and function induced by MLN2238 was at least partly due to induction of T-cell factor 4 (TCF4) transcriptional activity, as well as to activation of the unfolded protein response. A similar range of MLN2238 doses also markedly inhibited OC formation and resorption from human progenitors. Similarly to that described with bortezomib, MLN2238 treatment of human pre-OCs prevented RANKL-induced NF-κB activation, disrupted the integrity of the F-actin ring and also reduced the expression of the αVβ3 integrin, thus contributing to inhibition of OC function. MLN2238 was also able to overcome the growth advantage conferred to MM.1S-luc cells by co-culture with MSCs or OCs. Oral administration of MLN2238 in a mouse model of disseminated human MM decreased human RPMI-8226-luc tumor burden as assessed by diminished bioluminescence signal and decreased serum levels of Igλ secreted by RPMI-8226-luc cells. In addition, MLN2238 prevented tumor-associated bone loss with significant increases in femoral trabecular bone parameters as compared to vehicle control animals. Serum markers of bone turnover showed that MLN2238 inhibited bone resorption (decreased levels of CTX) while enhancing bone formation (increased levels of P1NP). Conclusion: MLN2238 in vitro was capable of promoting osteoblastogenesis and OB activity as well as of inhibiting OC formation and function to an extent similar to bortezomib. In a disseminated human MM mouse model, orally administered MLN2238 showed anti-resorptive and bone-anabolic effects in addition to its anti-tumor properties. Given the thus far available data on the preclinical safety and favorable pharmacologic properties of MLN2238, it is conceivable that MLN9708, the clinical formulation of this proteasome inhibitor, may also achieve bone benefits in myeloma patients. Disclosures: Berger: Millennium Pharmaceuticals, Inc.: Employment. San-Miguel:Millennium Pharmaceuticals, Inc.: Consultancy.

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p53 regulates the cardiac transcriptome

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1621436114 ◽

2017 ◽

Vol 114 (9) ◽

pp. 2331-2336 ◽

Cited By ~ 47

Author(s):

Tak W. Mak ◽

Ludger Hauck ◽

Daniela Grothe ◽

Filio Billia

Keyword(s):

Cardiac Tissue ◽

Acute Stress ◽

Gene Expression Signature ◽

Gene Clusters ◽

Biomechanical Stress ◽

Adult Mice ◽

Genome Wide ◽

Pathway Analyses ◽

And Function

The tumor suppressor Trp53 (p53) inhibits cell growth after acute stress by regulating gene transcription. The mammalian genome contains hundreds of p53-binding sites. However, whether p53 participates in the regulation of cardiac tissue homeostasis under normal conditions is not known. To examine the physiologic role of p53 in adult cardiomyocytes in vivo, Cre-loxP–mediated conditional gene targeting in adult mice was used. Genome-wide transcriptome analyses of conditional heart-specific p53 knockout mice were performed. Genome-wide annotation and pathway analyses of >5,000 differentially expressed transcripts identified many p53-regulated gene clusters. Correlative analyses identified >20 gene sets containing more than 1,000 genes relevant to cardiac architecture and function. These transcriptomic changes orchestrate cardiac architecture, excitation-contraction coupling, mitochondrial biogenesis, and oxidative phosphorylation capacity. Interestingly, the gene expression signature in p53-deficient hearts confers resistance to acute biomechanical stress. The data presented here demonstrate a role for p53, a previously unrecognized master regulator of the cardiac transcriptome. The complex contributions of p53 define a biological paradigm for the p53 regulator network in the heart under physiological conditions.

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Dysregulation of TLR9 in neonates leads to fatal inflammatory disease driven by IFN-γ

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1911579117 ◽

2020 ◽

Vol 117 (6) ◽

pp. 3074-3082

Author(s):

Alison G. Stanbery ◽

Zachary R. Newman ◽

Gregory M. Barton

Keyword(s):

Inflammatory Disease ◽

Autoinflammatory Disease ◽

In Vivo Model ◽

Type I ◽

Receptor Signaling ◽

Adult Mice ◽

Specific Receptors ◽

Ifn Γ ◽

Low Levels

Recognition of self-nucleic acids by innate immune receptors can lead to the development of autoimmune and/or autoinflammatory diseases. Elucidating mechanisms associated with dysregulated activation of specific receptors may identify new disease correlates and enable more effective therapies. Here we describe an aggressive in vivo model of Toll-like receptor (TLR) 9 dysregulation, based on bypassing the compartmentalized activation of TLR9 in endosomes, and use it to uncover unique aspects of TLR9-driven disease. By inducing TLR9 dysregulation at different stages of life, we show that while dysregulation in adult mice causes a mild systemic autoinflammatory disease, dysregulation of TLR9 early in life drives a severe inflammatory disease resulting in neonatal fatality. The neonatal disease includes some hallmarks of macrophage activation syndrome but is much more severe than previously described models. Unlike TLR7-mediated disease, which requires type I interferon (IFN) receptor signaling, TLR9-driven fatality is dependent on IFN-γ receptor signaling. NK cells are likely key sources of IFN-γ in this model. We identify populations of macrophages and Ly6Chi monocytes in neonates that express high levels of TLR9 and low levels of TLR7, which may explain why TLR9 dysregulation is particularly consequential early in life, while symptoms of TLR7 dysregulation take longer to manifest. Overall, this study demonstrates that inappropriate TLR9 responses can drive a severe autoinflammatory disease under homeostatic conditions and highlights differences in the diseases resulting from inappropriate activation of TLR9 and TLR7.

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Carfilzomib-Induced Cardiotoxicity in an In Vivo Model of Aging

Blood ◽

10.1182/blood-2020-136888 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 18-18

Author(s):

Panagiotis Efentakis ◽

Garyfallia Psarrakou ◽

Panagiota-Efstathia Nikolaou ◽

Michael Chatzistefanou ◽

Eleni-Dimitra Papanagnou ◽

...

Keyword(s):

Board Of Directors ◽

Research Funding ◽

The Elderly ◽

In Vivo Model ◽

Control Group ◽

Myocardial Tissue ◽

Advisory Committees ◽

Aged Mice ◽

Young Mice

Introduction: Carfilzomib (Cfz) is an approved irreversible proteasome inhibitor for the treatment of patients with relapsed/refractory multiple myeloma (R/R MM). Despite remarkable efficacy in R/R MM, Cfz clinical use is hampered by the incidence of cardiotoxicity. Age is recognized as an independent factor for the manifestation of cardiac failure and cardiovascular events. We have previously established a translational in vivo model of Cfz-induced cardiotoxicity, in which metformin (Met) had a potent prophylactic therapy, as it restored AMP-activated kinase α (AMPKα)-dependent autophagy in the myocardium of young mice, which had been inhibited by carfilzomib treatment (Efentakis P et al. Blood. 2019;133(7):710-723). Taking into consideration that MM is primarily a disease of the elderly, we sought to investigate whether our previous findings in young mice could be recapitulated in an aging in vivo model. Methods: Ten young C57Bl/6 mice (12-14 weeks of age) and thirty aged C57Bl/6 mice (15-17 months of age) were randomly assigned as follows: (i) Control group [Normal Saline (N/S) 0.9%, n=6]; (ii) Cfz group (8 mg/kg, n=6); (iii) Met group (140mg/kg, n=6); (iv) Cfz+Met group (8 mg/kg and 140 mg/kg respectively, n=6). N/S and Cfz were administered intraperitoneally on alternate days, while Met was administered per os daily for 7 days. At baseline and at the end of the experiments, mice were anesthetized with isoflurane (2% in 100% O2) and underwent echocardiography in order to investigate cardiac contractility markers (fractional shortening, FS%) and carotid plasticity markers (pulsatility index, PI% and resistance index, RI%). Subsequently mice were sacrificed for blood and myocardial tissue collection. Peripheral blood mononuclear cell (PBMCs), isolated from the whole blood, as well as myocardial tissue underwent proteasome activity assessment. Snap-frozen myocardial tissue underwent molecular immunoblotting analysis for the investigation of the molecular signaling. Results: Aged mice did not show any decreased proteasomal activity neither in the PBMCs or in the myocardium versus young C57Bl/6 mice. Cfz decreased proteasomal activity both in the PBMCs and the myocardium independently of Met administration. Aged mice presented a significant reduction of the FS% compared to the young mice at baseline, which represents an already established cardiac dysfunction in the elderly mice (mean FS%±SD: 37.40±1.6 vs. 45.62±0.8, respectively, p<0.005). In compliance with our previous findings in young C57Bl/6 mice, Cfz deteriorated the already present cardiac dysfunction in aged mice versus controls (mean FS%±SD: 28.2±1.8 vs. 37.8±1.8, respectively, p<0.05). Cfz+Met co-administration in elderly mice showed a marginal increase in terms of FS% compared to Cfz only treated mice (mean FS%±SD: 32.60±2.1 vs. 28.2±1.8, respectively), while FS% in the Cfz+Met group continued to be lower compared to control group (32.60±2.1 vs. 37.8±1.8). Assessment of the carotid stiffness revealed that Cfz sub-acute treatment led to a decrease in PI% compared to controls (p<0.05), while no changes in RI% were observed among groups, indicating a Cfz-induced vascular hypo-contraction in the elderly mice. Molecular analysis of the myocardial tissues showed that Cfz led to a decreased AMPKα and Protein Kinase B (Akt) phosphorylation, while Met restored AMPKα phosphorylation and increased endothelial nitric oxide synthase (eNOS) and Akt expression in the Cfz+Met co-administration group. Conclusion: Cfz induced cardiotoxicity in this aged murine model, in accordance with our previous findings in the young mice. Additionally, sub-acute Cfz treatment leads to a decrease in pulsatility capacity of the vessels, possible leading to vascular hypo-contraction in vivo. Co-administration with metformin exerts cardioprotection, even in the elderly mice, in an AMPKα-dependent manner. The latter is of great clinical significance as it further supports the translational value of metformin as a potent prophylactic therapy against Cfz-induced cardiotoxicity. Disclosures Efentakis: Amgen: Research Funding. Kastritis:Amgen: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Genesis Pharma: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria. Dimopoulos:BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Personal fees; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Personal fees, Research Funding, Speakers Bureau; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Personal fees, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Personal fees, Speakers Bureau; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Personal fees, Research Funding, Speakers Bureau. Andreadou:Amgen: Research Funding. Terpos:Genesis: Honoraria, Other: Travel expenses, Research Funding; Celgene: Honoraria; Sanofi: Honoraria; BMS: Honoraria; Amgen: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Takeda: Honoraria, Other: Travel expenses, Research Funding.

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Cells exhibiting strong p16INK4a promoter activation in vivo display features of senescence

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1818313116 ◽

2019 ◽

Vol 116 (7) ◽

pp. 2603-2611 ◽

Cited By ~ 43

Author(s):

Jie-Yu Liu ◽

George P. Souroullas ◽

Brian O. Diekman ◽

Janakiraman Krishnamurthy ◽

Brandon M. Hall ◽

...

Keyword(s):

Low Frequency ◽

Transcript Abundance ◽

Serial Passage ◽

In Vivo Model ◽

Promoter Activation ◽

Adult Mice ◽

Isolation And Characterization ◽

Secretory Phenotype

The activation of cellular senescence throughout the lifespan promotes tumor suppression, whereas the persistence of senescent cells contributes to aspects of aging. This theory has been limited, however, by an inability to identify and isolate individual senescent cells within an intact organism. Toward that end, we generated a murine reporter strain by “knocking-in” a fluorochrome, tandem-dimer Tomato (tdTom), into exon 1α of the p16INK4a locus. We used this allele (p16tdTom) for the enumeration, isolation, and characterization of individual p16INK4a-expressing cells (tdTom+). The half-life of the knocked-in transcript was shorter than that of the endogenous p16INK4a mRNA, and therefore reporter expression better correlated with p16INK4a promoter activation than p16INK4a transcript abundance. The frequency of tdTom+ cells increased with serial passage in cultured murine embryo fibroblasts from p16tdTom/+ mice. In adult mice, tdTom+ cells could be readily detected at low frequency in many tissues, and the frequency of these cells increased with aging. Using an in vivo model of peritoneal inflammation, we compared the phenotype of cells with or without activation of p16INK4a and found that tdTom+ macrophages exhibited some features of senescence, including reduced proliferation, senescence-associated β-galactosidase (SA-β-gal) activation, and increased mRNA expression of a subset of transcripts encoding factors involved in SA-secretory phenotype (SASP). These results indicate that cells harboring activation of the p16INK4a promoter accumulate with aging and inflammation in vivo, and display characteristics of senescence.

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An In Vivo Model To Assess Biomaterial Thromboresistance

10.1055/s-0038-1652255 ◽

1981 ◽

Author(s):

R Rodvien ◽

J Robinson ◽

R R Mitchell ◽

P Litwak

Keyword(s):

Minimally Invasive ◽

Axial Length ◽

Carotid Arteries ◽

Catheter Placement ◽

In Vivo Model ◽

Clinical Use ◽

Excellent Method

A minimally invasive model has been developed in which control and test No.5 French catheters are passed retrograde from the lingual to the carotid arteries. Goats are given autologous lll In platelets 48 hours before catheter placement, and, after placement, scanned externally and continuously for 3 hours. Net radionuclide retention occurs for the first 30 to 90 minutes after which lll ln leaves the catheter. Simultaneously placed polyethylene (PE) catheters are non-interactive; the weight of thrombus recovered/cm and platelets/cm for 18 goats (36 catheters) is 19.8 mg ± 9.8 and 8.87 × 108 ± 7.5 respectively. Thrombus is evenly distributed along the axial length of the PE catheter, increasing effective catheter diameter by 44%. Correlation between recovered thrombus/cm catheter and net platelet retention/cm catheter is 0.924 for PE. Various catheters exposed to 0.2 mg/ml albumin or 4 mg/ml albumin retained albumin but accumulated equal amounts of thrombus and platelets as untreated PE catheters. Glutaraldehyde used on albuminated catheters also did not change thrombus and platelet retention. Less thrombus was recovered and less platelets retained by PVC, BiomerR and teflon than by PE catheters. This model provides an excellent method to evaluate different polymers fabricated as catheters, both to understand thrombus growth and dissolution, and to choose among catheters for clinical use.

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